Neonatal jaundice occurs in 60-70% of newborns. Severe hyperbilirubinemia can lead to neurological dysfunction and possibly death, and preterm infants are particularly susceptible to these complications. Current diagnostic techniques measure total serum levels of bilirubin (TSB), which are used as indicators for treatment with phototherapy. However, levels of free, unbound bilirubin (Bf) are thought to determine the risk of bilirubin neurotoxicity more accurately than TSB. Bf levels can be elevated by competition for binding sites with free fatty acids (FFA). Most FFA circulate bound to albumin, with only a small fraction present unbound in the aqueous phase (FFAu). FFA are an important element of nutrition, so prior to full enteral feeding most premature infants receive them as Intralipid, an intravenous fatty acid emulsion. Preliminary data suggest that FFAu can be markedly elevated in premature infants receiving Intralipid, most likely due to immature metabolism and storage. This may lead to lipid associated complications, including elevated Bf secondary to competition with bilirubin for albumin binding sites. The thresholds for initiation and duration of phototherap are not well established in preterm infants, leading to conflicting approaches based on TSB, birth weight and/or gestational age. Thus, although levels of Bf-the toxic compartment of circulating bilirubin-may be markedly elevated in infants receiving Intralipid, current approaches do not include direct measurement of Bf. Recently, we developed FFAu- and Bf-specific probes through a process of iterative mutations and high throughput screening of fluorescently labeled FFA binding proteins. We demonstrated that these probes can detect physiologic, nanomolar concentrations of Bf and FFAu in plasma. The proposed studies will utilize this new, innovative technology to investigate the hypothesis that 1) Intralipid infusion will increase FFAu and consequently Bf and the Bf/TSB ratio in preterm infants, and 2) premature infants treated with phototherapy during the first week of life (based on TSB levels) who are receiving Intralipid will have at least 20% higher levels of Bf than those not receiving Intralipid. Such findings would support the idea that TSB underestimates risk in Intralipid-treated infants, and that Bf may be a better determinant of the need for phototherapy. Larger studies will then be designed to test clinical efficacy and improved outcomes after managing hyperbilirubinemia in preterm infants based on direct measurements of Bf.